Roses (Rosa spp.) are widely cultivated as ornamental plants in both Korea and worldwide. However, black spot disease caused by Diplocarpon rosae (= Marssonina rosae) continues to result in considerable damage in commercial production. While investigating black spot disease of roses collected from 27 regions across Korea, fungal strains were isolated from abnormal leaves exhibiting black spot symptoms to examine their distribution and characteristics. Regardless of region or cultivar, all isolates exhibited similar morphology, characterized by dark brown to black acervuli on symptomatic rose leaves and hyaline, two-celled conidia. The morphological characteristics of the representative strain KNUF-24-DG, including conidial size (16.5–25.8 × 4.7–6.5 µm) and cultural characteristics on potato dextrose agar, were similar to those previously described for D. rosae. For phylogenetic analyses of the 27 fungal strains, the internal transcribed spacer (ITS) regions and large subunit (LSU) rRNA gene were amplified. The ITS region showed 97.7–100% similarity to D. rosae strains CBS 163.31, CFCC 6814, KACC 42437, and DKU017, and the LSU gene sequences exhibited 99.6% and 100% similarities with D. rosae CBS 163.31 and CBS 829.72, respectively, indicating high sequence similarity to D. rosae strains in both molecular markers. Based on cultural, morphological, and phylogenetic analyses, 27 fungal strains were identified as D. rosae.